1. Field of the Invention
In the reaction by the use of an immobilized lipase, such heterogeneous substrates as an oily substrate and a water-soluble substrate are caused by mass transfer to reach a lipase held fast in a particulate immobilizing carrier, undergo transformation into heterogeneous products, i.e. an oily product and a water-soluble product, by the lipase, and depart from the lipase again by mass transfer. This invention is directed to a method for performing the reaction advantageously by enabling the mass transfer of the continuous-phase substrates to proceed efficiently and continuously.
The lipase is an enzyme which acts upon ester bonds and, as such, finds utility in such reactions as ester hydrolysis, ester synthesis, and transesterification. It is also utilized for the optical resolution of racemic esters, acids, and alcohols. Thus, the lipase is expected to find extensive utility in various applications in the oil and fat industry, the pharmaceutical industry, and the foodstuff industry, for example.
2. Prior Art Statement
A method which, by the use of a column packed with a photolinkable gel entrapping an immobilized lipase, carries out the reaction by preparatorily mixing water and an oil by stirring and circulating the resultant oil-water mixture in the form of emulsion to the column has been known to the art [Y. Kimura et al., Eur. J. Appl. Microl. Biotechnol., 17, 107 (1983)]. Besides, a method which, by the use of an immobilized lipase column developed earlier by the inventors, carries out the reaction by continuously feeding into the column a water-soluble substrate via a middle upper stage and an oily substrate via a middle lower stage respectively and collecting continuously an oil product through the upper end of the column and a water-soluble product through the lower end thereof (Kosugi et al.; U.S. Pat. application Ser. No. 586,563, dated Mar. 6, 1984, now abandoned) and a method which, by the use of a multistage reaction vessel, effects the reaction by alternately repeating separation and mixture of an immobilized lipase, an oily substrate, and a water-soluble substrate thereby bringing the oily substrate and the water-soluble substrate into counterflow contact with the immobilized lipase (Kosugi et al.; Japanese Patent Public Disclosure SHO 63(1988)-59896) have been known to the art.
The known method which involves the circulation of a preparatorily emulsified mixture to the immobilized lipase column cannot be easily carried out in the form of a continuous operation because the emulsion particles are diffused inside the immobilizing carrier at a notably low speed and, therefore, the reaction solution must be circulated to the immobilized lipase column time and again. Moreover, this method is incapable of separately collecting the oily product and the water-soluble product continuously.
The method which involves the counterflow supply of the oily substrate and the water-soluble substrate to the immobilized lipase column is required to secure a flow path adapted for the oily substrate and the water-soluble substrate to be advanced as preparatorily mixed with each other so as to preclude the phenomenon of channeling, i.e. complete separation of the flow path of the oily substrate from that of the water-soluble substrate inside the column, and also is required to operate the column in such a manner that the feed rate of the substrates will be lower than the speed of separation between the water and the oil inside the column. Thus, this method is subject to numerous operational restrictions and, therefore, cannot easily make full use of the activity of the immobilized lipase.
The method which, by the use of the multistage reaction vessel, effects the reaction by alternately repeating separation and mixing of the immobilized lipase, the oily substrate, and the water-soluble substrate thereby bringing the oily substrate and the water-soluble substrate into counterflow contact with the immobilized lipase primarily consists in a batchwise operation of the multistage reaction vessel. For this method to produce a continuos reaction and permit continuous collection of the oily product and the the water-soluble product, therefore, the reaction vessel requires a complicated piping system and the operation of this piping system requires a complicated control. Further, the mixing of the three components inevitably entails formation of a fine emulsion, rendering it difficult to separate the oil from the water. If the separation and the mixing were carried out in two separate reaction vessels to preclude the difficulty of the separation, the operation would necessitate a separation time in addition to the reaction time and could hardly be called an efficient method.
The inventors have already developed an immobilized lipase capable of retaining the activity thereof even in the presence of a higher fatty acid and have demonstrated that this immobilized lipase permits continuous protracted use (U.S. Pat. application Ser. No. 586,563, dated Mar. 6, 1984). Since this immobilized lipase uses as its carrier an ion exchanger specifically developed with a view to lowering the head loss of the liquid inside the column. Thus, this immobilized lipase is not suitable for the fluidized bed to be used in the method of the present invention.
As an example of the immobilized lipase used in the form of a fluidized bed, the use of a lipase immobilized in stainless steel beads of a large specific gravity has been reported. In the reported experiment, hydrolysis of a tributyrin emulsion is carried out in a fluidized bed reactor keeping the immobilized lipase in a floating state therein [R. B. Liberman et al.; biotechnol. Bioeng., 17, 1401 (1975)].
The above mentioned method causes the emulsion of substrates to flow up and circulate through a mass of fine beads of immobilized lipase having a large specific gravity. It is, therefore, not free from the influence of the resistance offered to the diffusion of the emulsion particles inside the carrier, is incapable of producing a perfect continuous reaction, and is unable to effect separate collection of the oily product and the water-soluble product. If the immobilized lipase of a large specific gravity is used in the method of the present invention, the possibility ensues that the immobilized lipase will be discharged in conjunction with the water-soluble product of a large specific gravity.